Molecular Brain Research 103 (2002) 91–105 www.elsevier.com / locate / bres Research report Molecular, functional, and genomic characterization of human KCC2, q the neuronal K–Cl cotransporter a ,1 a,b,c,1 c a a ´ Luyan Song , Adriana Mercado , Norma Vazquez , Qizhi Xie , Reshma Desai , a c a,b, * Alfred L. George Jr. , Gerardo Gamba , David B. Mount a Department of Medicine, Nashville VA Medical Center and Vanderbilt University Medical Center, Nashville, TN 37232, USA b Program in Membrane Biology, Department of Medicine, West Roxbury VA Medical Center and Brigham and Women’ s Hospital, Harvard Medical School, Room 542, 77 Avenue Louis Pasteur, Boston, MA 02115, USA c ´ ´ ´ ´ Molecular Physiology Unit, Instituto Nacional de Ciencias Medicas y Nutricion Salvador Zubiran and Instituto de Investigaciones Biomedicas, ´ ´ Universidad Nacional Autonoma de Mexico, Tlalpan 14000, Mexico City, Mexico Accepted 6 March 2002 Abstract The expression level of the neuronal-specific K–Cl cotransporter KCC2 (SLC12A5) is a major determinant of whether neurons will respond to GABA with a depolarizing, excitatory response or a hyperpolarizing, inhibitory response. In view of the potential role in human neuronal excitability we have characterized the hKCC2 cDNA and gene. The 5.9 kb hKCC2 transcript is specific to brain, and is induced during in vitro differentiation of NT2 teratocarcinoma cells into neuronal NT2-N cells. The 24-exon SLC12A5 gene is on human chromosome 20q13, and contains a polymorphic dinucleotide repeat within intron 1 near a potential binding site for neuron-restrictive 2 86 1 silencing factor. Expression of hKCC2 cRNA in Xenopus laevis oocytes results in significant Cl -dependent Rb uptake under isotonic conditions; cell swelling under hypotonic conditions causes a 20-fold activation, which is blocked by the protein phosphatase inhibitor calyculin-A. In contrast, oocytes expressing mouse KCC4 do not mediate isotonic K–Cl cotransport but express much higher absolute transport activity than KCC2 oocytes under hypotonic conditions. Initial and steady state kinetics of hKCC2-injected oocytes were 1 2 performed in both isotonic and hypotonic conditions, revealing K s for K and Cl of 9.361.8 mM and 6.860.9 mM, respectively; both m 2 2 affinities are significantly higher than KCC1 and KCC4. The K for Cl is close to the intracellular Cl activity of mature neurons, as m befits a neuronal efflux mechanism.  2002 Elsevier Science B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters and receptors Topic: GABA Keywords: GABA 1. Introduction Abbreviations: BAC, bacterial artificial chromosome; CIP, cation– chloride cotransporter interacting protein; DIDS, 4,4-diisothiocyanostil- GABA and glycine are the major inhibitory neuro- bene-2,2-disulfonic acid; DIOA, R( 1)-[(2-n-butyl-6,7-dichloro-2- cyclopentyl-2,3-dihydro-1-oxo-1-H-indenyl-5-yl)-oxy]acetic acid; EST, transmitters in the mature mammalian brain, however there expressed sequence tag; GABA, g-aminobutyric acid; HEK-293, human is a brief developmental window in the first week of embryonic kidney cell line; HUVEC, human umbilical vein endothelial postnatal life during which they evoke an excitatory cells; KCC, K–Cl cotransporter; NEM, N-ethylmaleimide; NKCC1 / response. The activation of glycine and GABA receptors A NKCC2, Na–K–2Cl cotransporter; SLC12A5, solute carrier family 12, in immature neurons thus causes membrane depolarization member 5 (HUGO / Human Genome Organization nomenclature for 2 2 KCC2); STS, sequence tagged site; TM, transmembrane segment due to the efflux of Cl through ligand-gated Cl chan- q The hKCC2 sequence has been deposited in Genbank under accession nels, resulting in neuronal excitation [46]. The subsequent 21 number AF208159. influx of Ca , via both voltage-dependent and NMDA- *Corresponding author. Tel.: 11-617-525-5876; fax: 11-617-525- 21 gated Ca channels [12,37], has significant trophic [41] 5830. and neurodevelopmental consequences [2,13,29]. In con- E-mail address: dmount@rics.bwh.harvard.edu (D.B. Mount). 2 1 Equal contribution. trast, intracellular Cl in mature neurons is much lower, 0169-328X / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0169-328X(02)00190-0